CN1168377C - Method of quick-acting cotton transgenosis - Google Patents
Method of quick-acting cotton transgenosis Download PDFInfo
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- CN1168377C CN1168377C CNB011310871A CN01131087A CN1168377C CN 1168377 C CN1168377 C CN 1168377C CN B011310871 A CNB011310871 A CN B011310871A CN 01131087 A CN01131087 A CN 01131087A CN 1168377 C CN1168377 C CN 1168377C
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Abstract
The present invention belongs to the field of cotton transgenic breeding technology, which is characterized in that tissue cultivation is utilized to induce a hypocotyledonary axis of cotton to obtain callus; embryonic callus is obtained by successive cultivation so as to obtain the embryonic callus to be used as an agroinfection receptor; the resistance callus is obtained by selecting and sieving culture media, and then plants are generated; the aging-resistance transgenic cotton is obtaind by molecular detection. Compared with the prior art, the present invention can greatly enhance the conversion efficiency of cotton, greatly shortens the conversion cycle, and greatly increases conversion bodies, and furthermore, the transgenic plants can be obtained in 6 months. The aging-resistance transgenic (ipt) cotton, such as Coker 201 (ipt), Ekang No. 3 (ipt) and YZ1 (ipt), is obtained.
Description
Technical field
The invention belongs to the new plant technical field, be specifically related to the cotton transgenic breeding technical field.
Background technology
At present the genetic transformation technology of cotton mainly contains following several class methods: the one, utilize the hypocotyl (Umbek of Agrobacterium tumefaciens mediated cotton, et al, the genetic transformation of cotton, Bio/Teconology, 1987,5:263-267), the 2nd, utilize pollen tube passage method (Huang Junqi etc., external source sea-island cotton DNA causes the variation of upland cotton proterties, Acta Genetica Sinica, 1981,8 volumes, 1 phase 56-62,) converting cotton, the 3rd, (microprojectile bombardment methods obtains transgene cotton to particle gun bombardment callus converting cotton for Finer, et al, Plant Cell Rep 1990,8:586-589).The problem of utilizing Agrobacterium tumefaciens mediated cotton hypocotyl mainly to exist has: the one, and the time that needs from the hypocotyl to the plant regeneration is longer, generally take more than 8 months, the 2nd, utilize Agrobacterium to infect hypocotyl, can influence the acquisition of callus, the conversion piece of Huo Deing is also less simultaneously.Utilize the pollen tube passage method converting cotton then to exist the transformation frequency low repeatability bad, and transformant is also limited.Then chimera is more to utilize gene bombardment callus, and the foreign gene multicopy gene silencing that easily causes in the majority that transforms, and genetic stability is poor, and the experimental cost height.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, utilize Agrobacterium tumefaciens mediated embryo callus subculture converting cotton, both combined the advantage of Agrobacterium-mediated Transformation, it is long to have overcome the cycle again, the shortcoming that transformant is few, thereby can improve the efficient of cotton transgenic breeding.
The present invention is achieved through the following technical solutions:
A kind of method that obtains transgenic cotton plant is characterized in that, utilizes tissue culture inducing cotton hypocotyl to obtain callus, obtains embryo callus through successive transfer culture; Utilize the pre-embryo callus of cultivating 12 days to carry out common cultivation as the agroinfection acceptor then, described cultivation temperature altogether is 16-26 ℃, and Agrobacterium bacterial concentration OD value is 0.3-0.5; After cultivating 48 hours altogether, use 0.01%HgCl
2Handle embryo callus 5-10 minute of agroinfection and take off bacterium; Change on the selection medium that is added with the 50mg/L kanamycin, successive selection 2-3 time obtains resistant calli by selecting the medium screening again; This callus successive transfer culture is obtained complete regeneration plant on differential medium, obtain anti-ageing transgenic cotton plant through Molecular Detection.
Description of drawings
Fig. 1 is callus growth state diagram of the present invention;
Fig. 2 is a transgenosis callus PCR testing result of the present invention; Among the figure, 1:Marker (GenrulerTM 100BP DNA ladder); 2-11: transform the callus clone; 12: negative control; 13: plasmid.
Fig. 3 is a transgenic line Southern results of hybridization of the present invention, and DNA cuts through Xab I enzyme among the figure, with SAG12 gene-amplified fragments 560bp probe of marking.Swimming lane 1:Mark (λ DNA/HindIII); 2: negative control; 3: positive control; 4-7: transformed plant
The invention will be further described below in conjunction with Figure of description:
1, the activation of Agrobacterium: bacterial strain is after the bacterium colony screening, LB fluid nutrient medium activation 12 hours, then inoculate on the LB solid medium, 28 ℃ of dark cultivations two days, then it is scraped that (every liter contains 5g tryptone, 5gNaCl, 0.1gMgSO into the acetosyringone that is added with 20mg/L (AS) MGL4.7H
2O、0.25g KH
2PO
4, 5g sweet mellow wine, 1.16g Sodium Glycinate) in the culture medium, 240rpm shaken cultivation 2 hours, being diluted to bacteria containing amount with the MSB fluid nutrient medium is 108～10
10About individual/L. (OD600＝0.4～0.8)
2, get active high cotton embryo callus subculture, put into the Agrobacterium suspension bacteria liquid for preparing, soak 5-10min, vacuumize simultaneously once, shake bottle in the process for several times, fall dried bacterium liquid, blot the surface with sterilization blotting paper, then access is total in the culture medium, places 19-21 ℃, the dark cultivation two days, this moment is at the visible a small amount of bacterium liquid of the part of culture medium contact explant.
3, the screening of resistant material: will cultivate altogether processing the additional 500mg/L cephalosporin of callus (or utilize mercury chloride to take off bacterium, as described later) and on the kanamycins of the 50mg/L selection culture medium, subculture 2-3 time; Afterwards with kalamycin resistance callus subculture and the cultivation that suspends.
4, plant regeneration is transferred to differential medium with embryoid, behind the regeneration seedling regrowth is moved into in the seedling medium.
5, the acquisition of the evaluation of transgenic seedling and transfer-gen plant: smear newborn blade with the solution of kanamycin 2% earlier and make Preliminary screening, remake PCR and detect, carry out Southern at last and analyze.
Utilize above-mentioned steps, the present invention has successfully obtained anti-ageing transgene cotton Coker201 (ipt), and Hubei Province resists No. 3 (ipt) and YZ
1(ipt).YZ wherein
1Be a kind of super chicken foot leaf upland cotton strain.
Effect of the present invention is:
1, utilize embryo callus can increase substantially the cotton transformation efficiency as infecting acceptor, be additive method can not compare.
2, the cycle of Zhuan Huaing shortens greatly, generally obtains transfer-gen plant needs 1 year or longer time, and the present invention can obtain transfer-gen plant in 6 months, and transformant can increase considerably.Can insert transform T-DNA like this and come true, be that technology platform has been prepared in the research of functional genome thereby can create a large amount of insertion mutant.
3, utilize Agrobacterium tumefaciens mediated embryo callus subculture in half a year, can access thousands of transformant, under same workload, the transformant efficient of taking in 1 year than the mediation hypocotyl is high about 200 times, and is higher about 400 times than particle bombardment and pollen tube mediated method.
4, utilize low concentration HgCl
2Handle, can disposablely remove Agrobacterium, and embryo callus subculture still can recover growth, not only solve other antibiotic and repeatedly suppressed the later stage differentiation that growth of Agrobacterium causes influencing callus, and saved expensive antibiotic, have certain economic and be worth.
Embodiment
Embodiment 1:
Utilize Agrobacterium tumefaciens mediated embryo callus to transform the isopentenyl transferase genes cotton.Utilize the method that isopentenyl transferase genes is changed over to acceptor cotton Coker201, Hubei Province anti-No. 3 and YZ
1Kind obtains transgenic regenerated plant, and concrete steps are as follows:
(1) transformation receptor State Selection:
The physiological status of callus is extremely important for efficient conversion.Generally speaking, meristematic tissue and the most responsive to infecting of Agrobacterium with enlivening fissional explant, promptly growing and dividing vigorous cell is the competence of accepting foreign DNA.But callus day growth amount is the split speed of reacting cells to a certain extent.Select the consistent embryo callus 0.3g of dispersion, color cadmium yellow naturally of growth to change in the D1 differential medium, establish 5 processing altogether, 3 repetitions are established in each processing, the callus fresh weight that claimed 1 processing every 3 days, callus growth rate (g/d)=(callus amount of growth-callus primary quantity)/growth fate.
When subculture in the time of 12 days, callus growth rate the fastest (as shown in Figure 1).Therefore subculture 10-12 days be in the graininess of enlivening splitting status, faint yellow, loose callus is the good receptor that transforms.Result of the test also proves, transformation efficiency (62.8%) height of 10 days callus transformation efficiency (80.3%) of subculture than 20 days.
(2) bacterial concentration is to the influence of conversion ratio:
With regard to the transient expression effect, high more its transformation efficiency of bacterial concentration is also high more, but the too high difficult inhibition of Agrobacterium in incubation of bacterial concentration, pollute callus (as shown in table 1) easily once again, and the Agrobacterium excessive concentration is easy to generate a large amount of noxious materials and causes the death of recipient cell, therefore be not that the Agrobacterium bacterial concentration is high more, then the kanamycin-resistant callus tissue rate is high more.Experimental result (as shown in table 1) shows that also the bacterium liquid of 0.3-0.5 O.D. concentration infects callus and can obtain higher kanamycin-resistant callus tissue rate (as shown in table 1).
The influence of table 1 bacterial concentration antagonism callus rate
| Bacterial concentration (OD value) | Handle callus piece number (individual) | Pollute callus piece number (individual) | Kanamycin-resistant callus tissue piece number (individual) | Kanamycin-resistant callus tissue percentage (%) | Cultivate back transient expression effect altogether |
| 0.1 | ?80 | ?1 | ?13 | ?16.25 | Low |
| 0.3 | ?150 | ?5 | ?117 | ?78.2 | High |
| 0.5 | ?170 | ?18 | ?136 | ?80 | High |
| 1.0 | ?90 | ?69 | ?15 | ?16.67 | 100％ |
| 1.5 | ?110 | ?83 | ?19 | ?17.27 | 100％ |
Annotate: pollute callus piece number and be meant the callus that Agrobacterium is not restrained and pollute once again.
(3) screening of resistant calli
After cultivating altogether, adopt HgCl
2Handle, can thoroughly eliminate Agrobacterium (being shown in Table 2).Utilize HgCl
2It is feasible replacing expensive cephalosporin or carbenicillin, and it is clean to take off bacterium, returns bacterium later on seldom again, is a kind of microbial inoculum that takes off preferably.
Table 2 utilizes HgCl
2Take off the recovery effects of bacterium and callus
The selection of resistant calli is one of key of whole transformation experiment, and effectively screening not only can reduce losing of transformant, and can alleviate the workload of later stage during Molecular Detection greatly.
Callus is transferred on the selection medium, and per three all subcultures once.Callus has just been transferred to when selecting on the medium, and most of callus all have a browning, but chimeric yellowish callus on a small quantity in the callus of sending out brown.Therefore the first round is selected generally not eliminate.When second takes turns selection, eliminate the callus piece of complete brownization.And the clone that will breed carries out carrying out liquid selective 15 days (Km is 100mg/L) after third round selects.When suspension culture each cell all with select medium in high concentration Km fully contact, can strictly eliminate chimera like this.
Therefore former take turns screening should not be tight excessively.Carry out strictness screening back when callus to be transformed is bred into the clone and just can indiscriminately press, help the embryoid seedling differentiation like this.
(4) the Molecular Detection result of resistant calli DNA
Fig. 2 is the result with the amplification of promotor SAG12 special primer.As can be seen from Figure 2, unconverted contrast does not have amplified band, and 10 transformed clones and plasmid all amplify the special band of an about 560bp of size, illustrate that external source SAG12 gene has been integrated in the genome of cell.The result of Southern (as shown in Figure 3) has further proved the conclusion of pcr analysis, and 4 clones callus and plasmids all have positive signal to occur, and contrast does not have.
Claims (1)
1, a kind of method that obtains transgenic cotton plant is characterized in that, utilizes tissue culture inducing cotton hypocotyl to obtain callus, obtains embryo callus through successive transfer culture; Utilize the pre-embryo callus of cultivating 12 days to carry out common cultivation as the agroinfection acceptor then, described cultivation temperature altogether is 16-26 ℃, and Agrobacterium bacterial concentration OD value is 0.3-0.5; After cultivating 48 hours altogether, use 0.01%HgCl
2Handle embryo callus 5-10 minute of agroinfection and take off bacterium; Change on the selection medium that is added with the 50mg/L kanamycin, successive selection 2-3 time obtains resistant calli by selecting the medium screening again; This callus successive transfer culture is obtained complete regeneration plant on differential medium, obtain anti-ageing transgenic cotton plant through Molecular Detection.
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|---|---|---|---|
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| CNB011310871A CN1168377C (en) | 2001-09-20 | 2001-09-20 | Method of quick-acting cotton transgenosis |
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| CN1168377C true CN1168377C (en) | 2004-09-29 |
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| CN103748223B (en) * | 2012-08-13 | 2016-11-09 | 创世纪种业有限公司 | One Cotton Gossypii prenyltransferase and encoding gene thereof and application |
| CN103070067A (en) * | 2013-02-05 | 2013-05-01 | 中国农业科学院棉花研究所 | Gene gun living quick transformation method of cotton |
| CN106258953A (en) * | 2016-07-18 | 2017-01-04 | 运城学院 | A kind of processing method of inducing cotton wound healing fast-growth |
| CN111321164A (en) * | 2020-03-12 | 2020-06-23 | 山西省农业科学院棉花研究所 | Method for reducing cotton transgenic material pantoea |
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